PROJECT SUMMARY Central aspects of the innate immune response are shared between Drosophila and mammals, but despite this extraordinary conservation, natural populations of both organisms harbor considerable genetic variability in efficacy of the immune response. Much of the functional variation results from differences in the transcriptional dynamics of the immune response. Evolution has tuned both positive and negative regulation of the immune system for robust control even in the presence of environmental and genetic perturbations. The first Specific Aim of this proposed project is to use naturally occurring variation in D. melanogaster to reverse engineer the cis- and trans-regulatory components that control transcription of the innate immune response. Both pyrosequencing and RNA-seq will be applied to F1 hybrid lines from the Drosophila Genetic Reference Panel before and after infection to obtain a quantitative profile of expression dynamics. GRO-seq of fat body tissue in phenotypically extreme lines will specify the role of paused polymerase in both the speed of response to septic injury and the precision of the transcriptional regulation. Network reconstruction and an ODE dynamical model of the network will be fitted using differences causes by genetic polymorphism among lines as perturbations to the gene regulatory network. The models will seek to reveal heretofore cryptic feedback loops and other regulatory constraints. The second Aim is to establish and dissect the role of microRNAs in regulating the innate immune response. High-throughput sequencing of libraries of short RNA molecules will be analyzed at various times before, during, and after infection to establish the time-course of microRNA expression over an experimental infection. Transcript targets of immune-responsive microRNAs will be computationally inferred and functionally validated. Variation among DGRP lines in the microRNA response will be joined with the RNA-seq and GRO-seq data obtained under Aim 1 to determine the impact of miRNA regulation on immune-related gene expression. Tests of association will be performed to identify segregating polymorphisms in the miRNA genes and their targets that may result in differences in expression dynamics. The third Aim is to define the kinetics of innate immune system shutdown after infectious threat is eliminated, and to assess whether the rapid shutdown is driven by the physiological or autoimmune cost of inappropriate immune system activity. Dynamics of transcript decay and negative regulation of immune-activating pathways will be quantified. The correlation of shutdown kinetics with fitness traits such as reduced fecundity and lifespan will be tested under the hypothesis that genetic lines which exhibit excessively slow shutdown dynamics incur fitness costs. Ultimately this project seeks to determine how evolutionary modulation of the innate immune response has struck the critical balance between robust anti- pathogen defense and protection of self-tissue from autoimmunological damage.